Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
  • Y10T436/108331—Preservative, buffer, anticoagulant or diluent
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
  • Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/25625—Dilution

Definitions

• This invention relates to the field of agglutination assays to detect the binding of ligands, and particularly immunological binding (antigen and antibody binding) such as that involved in blood group serology ( "immunohematology” ) .
• Immunohematology requires the determination of blood cell compatibility between a blood donor and a patient recipient before a transfusion or organ transplant involving the patient. Blood cell compatibility is determined by the non-occurrence of an immunological reaction between antibodies contained in the blood serum of a patient and antigens present on blood cells from the donor .
• Blood group antigens are found on the surface of the red blood cells of every individual. These antigens, the products of inherited genes, exist in a unique combination in everyone except identical twins . Blood grouping is generally the process of testing red cells to determine which antigens are present and which are absent, normally utilizing antibodies to the antigen tested for. Additionally, when a person does not have a particular red cell antigen on his or her red blood cells, his or her serum may contain antibody to that antigen. Whether or not the antibody is present in the serum depends on whether the person' s immune system has been previously challenged by, and responded to, that specific antigen or something very similar to it. For example, a person whose red blood cells are Type A, i.e. having "A" antigens on the red cells, will have anti-B antibodies in his or her serum. Thus, if such a person is given type B blood, an immunological reaction will occur with possible serious clinical consequences.
• the general approach known as the "Landsteiner” method, has been to add a patient's red blood cells to a standard laboratory test tube containing a blood group antibody (such as Anti-A or Anti-B) mix to allow antibody/antigen binding reactions to take place, and then to centrifuge. If the antigen tested for is present, the antibody/antigen binding will have taken place resulting in agglutination of the patient's red blood cells.
• the test tube is manually shaken to dislodge the centrifuged button of "clumped" cells at the bottom. A subjective determination is then made as to whether or not the dislodged cells are "clumped", and to what extent.
• the reactants are incubated for a period of time sufficient to permit agglutination of the red blood cells, which occurs when antibodies against those antigens are present. Such incubation typically ranges from about 10 minutes to about 40 minutes in modern testing.
• the mixture is then centrifuged, and if agglutinated blood cells are present, such agglutinates are clearly visible at the bottom of the reaction vessel, thus indicating the presence of antibodies in the sample directed against the known antigens on the red blood cells . If no antibodies are present in the sample directed against the known antigens on the red blood cells, agglutination does not occur, and this is indicated by the absence of agglutinated red cells after centrifugation.
• Antibodies of the ABO blood grouping system are generally immunoglobulin M (IgM) . These antibodies have ten antigen binding sites per molecule.
• the IgM antibody is large enough to span the distance between red blood cells, so that when they are centrifuged, the cells will be bound together in a lattice "cell-antibody-cell-antibody” and will remain clumped together in agglutinates. For example, if anti-A is added to blood group A or blood group AB cells and the mixture is centrifuged, the cells will remain in clumps when resuspended. With the same antibody, group O and group B cells will resuspend as individual cells. Agglutination facilitated by IgM antibodies is termed direct agglutination.
• Anti-D antisera is now manufactured as monoclonal IgM or polyclonal IgG mixtures. These reagents can p enotype RBCs on immediate spin without the presence of high protein additives.
• the human IgG molecule was also chemically modified to unhinge the disulfide bond so it could have a larger span and act like a IgM molecule.
• IgG antibodies cannot easily span the distance between cells which tend to repel each other in a saline environment. Thus, IgG will bind to red cell antigens matching its specificity, but will not directly agglutinate such red cells as effectively as the larger IgM antibodies will. The presence of IgG antibody bound to a red cell is thus usually detected by the addition of anti-IgG which will cause the requisite agglutination, resulting in a lattice of "red cell-IgG/Anti-IgG/IgG-red cell" .
• Tests for IgG bound to red cells in vivo are called direct antiglobulin tests.
• Tests for IgG bound to red cells in vitro are called indirect antiglobulin tests.
• Such antiglobulin tests are also called "Coombs" tests.
• This indirect antiglobulin test is a blood test used to determine whether there are IgG antibodies in a patient's serum to specified antigens on the surface of red blood cells. In the Coombs test, serum is incubated in the presence of reagent red cells to allow the antibodies to bind to antigens on the surface of the red cells.
• IgG antibodies most often do not, by themselves, agglutinate the red cells, or only agglutinate them insufficiently to be detected visually by conventional techniques. Addition of a second antibody directed to human IgG is usually necessary to facilitate visible agglutination.
• a convenient gel test and method of detecting antibodies or antigens is contemplated in this invention, wherein complexes of carrier-bound antibodies with antigens or carrier-bound antigens with antibodies in aqueous medium are made optically visible as before described.
• the carrier is preferably a gel or polymer and the antigens or antibodies as the case may be are bound to the carrier surface.
• the gel test contemplated herein is the Anti-Human Globulin Anti-IgG (Rabbit) MTS Anti-IgG
• CAT agglutination technology
• the invention is not limited to such test system and method but may be used with other formats besides gel such as for example, test tube, slide, solid phase and column agglutination technology (CAT) systems and methods, the latter whether in column or cassette form such as for instance the BioVue SystemTM of Ortho-Clinical Diagnostic Systems, Inc., Raritan, NJ, and might be used in any immunohematology system that employs incubation of the antibody and red cell antigen, regardless of test method.
• CAT column agglutination technology
• the Direct test which does not normally employ incubation, is accomplished by the employment of a gel card containing microtubes each of which contain an antibody incorporated into the gel matrix, and wherein diluted patient RBCs are placed on top of the gel carrier.
• Anti-human globulin (anti-IgG) is present in the gel.
• the card is centrifuged, which accelerates the reaction, if any, between the antibody reagent on the gel and the patient blood cells containing antigen, and also urges any cells toward the bottom of the microtubes.
• the gel in the microtubes act as a filter, however, and resist or impede downward movement of the particles in the microtube.
• the nature and distribution of the particles in the microtube after centrifuging provides a visual indication of whether any agglutination reaction occurred in the microtube, and if so, of the strength of that reaction.
• red blood cells agglutinate.
• the size of the pellet of red blood cells at the bottom of the microtube, and the extent to which the agglutinates penetrate into the gel in the microtube, are both inversely related to the strength of the reaction between the reagent and the red blood cells.
• the instant invention is a method to reduce time to result in blood bank immunohematologic testing for tests that use incubation of the antibody and the red cell antigen.
• antigen-antibody reactions including red cell typing reactions needing a incubation step, in immunohematology are detected by the visible agglutination of red blood cells or the evidence of hemolysis at the completion of testing.
• Such a test is for example, the Indirect or Reverse test as before mentioned, wherein patient antibodies are detected in sample plasma or serum by agglutination with diluted reagent RBCs.
• patient sample plasma or serum is added to a microtube containing reagent RBCs that have been diluted in a low ionic strength solution (diluted MTS Diluent 2TM) , the card containing the microtube is incubated with agitation, followed by centrifugation and removal and reading of the card for agglutination.
• a low ionic strength solution diluted MTS Diluent 2TM
• the instant invention allows for a decreased time for incubation due to potentiation of the antibody-antigen reaction as discussed below.
• the antibody attaches to an antigen on the red blood cell.
• antigenic determinants on the red blood cell combine with the antigen-binding site of the antibody molecule.
• the combination of an antigen and antibody is a random pairing of the two molecules determined largely by chance.
• Several factors influence the probability for this collision of antigen and antibody (Blaney K, Howard P: Basic and Applied Concepts of Immunohematology, St Louis, 2000, Mosby, and Vengelen-Tyler V: Technical manual, ed 12, Bethesda, Md, 1996, American Association of Blood Banks) , and include the following:
• the relative serum to cell ratio (i.e., the ratio of antigen on the red blood cell to antibody in the serum) will influence the probability of antigen-antibody combinations.
• Increasing the amount of serum in testing increases the concentration of antibodies available for binding to the red blood cell antigens.
• the number of antigen sites available on a per red blood cell basis also contributes to the strength of the antigen-antibody reaction.
• Antigen receptor accessibility The position of an antigen relative to the lipid bilayer of the red blood cell membrane contributes to its accessibility to antibody molecules, particularly IgG molecules. If the steric hindrance is decreased, the antibody molecules have a greater opportunity of interaction with the antigenic determinants.
• the temperature of the reaction influences the first stage of the agglutination reaction.
• most antibodies with clinical relevance are IgG immunoglobulins, which optimally react at temperatures of 37°C.
• IgM antibodies are more reactive at lower temperatures, generally at or below room temperature. Providing the suitable temperature in the reaction enhances the sensitization step.
• Allowing adequate time for the combination of antigen and antibody to attain equilibrium is also a factor that enhances the first stage of the agglutination reaction.
• the optimal pH for hemagglutination is approximately pH 7.0. This pH is adequate for the majority of important red blood cell antibodies.
• the use of low ionic strength solution as a potentiator of agglutination is a common practice in blood bank testing. For example, use of a buffer of about 0.03M is most useful. See Low and Messiter, Vo2 Sang 1974, Vol. 26, p. 53. Use of MTS Diluent 2TM (Micro Typing Systems, Inc., Pompano Beach, FL) is preferred.
• All current commercial antibody detection test methodologies have the initial step of having the antigen (RBC) and the antibody (serum or plasma) incubated at 37°C for a period of time, between 10-40 minutes and typically 15 minutes.
• the instant invention provides for a significant reduction of time of incubation in an immunohematologic assay by employing continuous agitation and optionally, low ionic strength diluent. This reduction of test time can be realized no matter what specific test format is being used, whether test tube test, slide test, solid phase test system, microcolumn or microtube, or microplates, and regardless of matrix material, for instance, whether gel or glass bead is employed as matrix.
• Blood bank testing has maximized the use of the factors previously outlined to produce diagnostic tests with appropriate sensitivity and specificity.
• the manipulation or combination of any of these variables of antigen-antibody reactions in test systems can reduce the time to result in blood bank testing.
• the instant invention is directed to reduction in incubation time required by use of continuous agitation while incubating.
• the actual amount of reduction in time can vary with the factors enumerated hereinabove, such as the ionic strength of the diluent, the presence or absence of enhancement agents (such as bovine albumin, polyethylene glycol, or proteolytic enzyme) the red blood cell/serum or plasma ratio, the initial temperature of the test sample and reagents, etc.
• enhancement agents such as bovine albumin, polyethylene glycol, or proteolytic enzyme
• the present invention provides a method and device for reducing time to result in blood bank diagnostic testing, using agitation and in a preferred embodiment, a low ionic strength buffer.
• the invention can employ a device that is an incubation chamber and agitation block which can accommodate one or more containers, for example agglutination sample test tubes, slides or the ID-Micro Typing SystemsTM gel cards containing microtubes as discussed above.
• the container is centrifuged and the presence or absence of agglutinates detected.
• a method for reducing time to result in immunohematology assays comprising performing an Indirect test comprising:
• the sample is plasma or serum.
• the continuous agitation is provided by a mechanical agitation block or manually.
• the anti-IgG matrix comprises for example gel beads .
• the anti-IgG matrix is disposed in. a test tube or microtube of the ID-Micro Typing SystemTM.
• the antigen-positive RBCs in step (a) are admixed with a low ionic strength diluent such as for example a hypotonic buffered solution employing buffers commonly used in the art such as for example HEPES and TRIS.
• a low ionic strength diluent such as for example a hypotonic buffered solution employing buffers commonly used in the art such as for example HEPES and TRIS.
• the low ionic strength diluent is less than about 0.03M.
• the immunohematologic technology used comprises microtubes disposed in gel card, form, such as in the ID- Micro Typing SystemTM
• the invention in a preferred embodiment comprises a method for performing an Indirect test comprising:
• step (b) and (c) at 37°C with continuous agitation for 2-15 minutes ; (e) adding the incubated admixture of step (d) to the microtube and centrifuging t ie microtube; and (f) reading the result.
• the sample is plasma or serum.
• the continuous agitation is provided by a mechanical agitation block or it may be provided manually.
• the test red blood cells in step (b) are admixed with a low ionic strength diluent of less than about 0.03 M.
• column agglutination technology specifically the BioVueTM column (Ortho- Clinical Diagnostics, Inc., Raritan, NJ) using the methods of the current invention.
• the methods employed in BioVueTM are described in detail in EP 485,228, EP 755,719, EP 725,276, US Pat. Nos .
• a reaction vessel comprising a microcolumn having a separation region and an incubation region wherein the separation region contains a matrix for separating agglutinated cells from unagglutinated cells.
• the method involves the similar mechanism of detecting the presence or absence of antibodies or antigens, preferably blood group antibodies or antigens by (a) adding an antibody or antigen detecting reagent and a liquid patient sample possibly containing an antibody or antigen, to the matrix which permits movement of non-agglutinated reactants but does not permit movement of agglutinated reactants; (b) applying a force to the matrix, for example centrifugal force, to effect movement through the matrix; and (c) detecting the presence or absence of agglutinated reactants .
• a method for reducing time to result in immunohematology assays comprising: (a) providing a microcolumn containing an upper chamber and a lower chamber which contains a matrix for separating agglutinated from non-agglutinated cells; (bl) depositing reagent red cells and patient serum or plasma sample to the upper chamber of the microcolumn; or (b2) depositing a reagent antibody with known specificity for red cell antigen and a patient red cell sample; (c) incubating the microcolumn of (bl) or (b2) at 37°C with continuous agitation for 2-15 minutes ,- (d) centrifuging the microcolumn; and (e) reading the result.
• the continuous agitation is provided by a mechanical agitation block or manually.
• the red blood cells in step (b) may be admixed with a low ionic strength diluent of preferably less than about 0.03M.
• a low ionic strength diluent is MTS Diluent 2TM (Micro Typing Systems, Inc., Pompano Beach, FL) that has itself been diluted to less than about 0.03M.
• This invention describes a method that uses a device or combination of devices and methods, to significantly reduce the incubation time required to accomplish the antigen-antibody binding (sensitization stage) in IgG- dependent red blood cell antibody reactions that employ an incubation step.
• the device and method employs continuous agitation during incubation, with simultaneous heating, to decrease the incubation time. It changes the rate of an antigen-antibody reaction through the increased movement of molecules in the reaction milieu, which increases the surface area for collisions between appropriate antigenic determinants and antibodies (Kaplan L, Pesce A: Clinical chemistry: theory, analysis, and correlation, St Louis, 1984, Mosby) .
• This method can be used in combination with a low ionic strength red blood cell diluent, which may be used to dilute the antibody screening cells prior to testing.
• the agitation method may be used in all immunohematologic technologies and formats whether test tube test, slide test, solid phase test system, microcolumn or microtube, or microplates, and regardless of matrix material, for instance, whether gel or glass bead is employed as matrix:.
• the method may be conducted using any device that may take the form for example of a mechanical agitation "block” or may be accomplished by a manual shaking/agitation.
• the incubation may be accomplished through use of an electric incubator device or other means such as a water bath.
• the implementation of the agitation device in combination with the low ionic strength red blood cell diluent reduced the incubation time from 15 minutes to 2 minutes in the samples tested. This reduction is significant in the performance of an indirect antiglobulin test.
• the shortest incubation time for indirect antiglobulin testing is typically between 10- 15 minutes. In indirect antiglobulin testing the incubation period is included to allow sufficient antibody uptake on the red blood cells for detection with antiglobulin reagents.
• Such low ionic strength solutions include those as disclosed by Low and Messiter, ibid, and MTS Diluent 2TM (Micro Typing Systems, Inc., Pompano Beach, FL) .
• the incubation times can be reduced in the performance of indirect antiglobulin testing to 10-15 minutes. Consequently, an agitation-incubation device combined with a low ionic strength red blood cell diluent maximizes antibody uptake on red cells and can significantly reduce the incubation time in indirect antiglobulin testing procedures.
• Such low ionic strength solution is typically less than 0.03M concentration.
• the agitation-incubation device may consist of an incubation chamber that maintains an environment at a temperature of 37°C and an agitation block.
• the agitation block is powered by an external power source.
• the incubator used in the prototype model was a Thermolyne Model 142300 (Barnstead International, Dubuque, IA) .
• the agitation block can be adapted to any incubation chamber for accommodation in any system that applies the use of trie agitation block in a test platform.
• the agitation block is connected to the external power source, which controls the rate of mixing during incubation.
• the agitation block is designed to hold test tubes and/or cards, and can be physically removed from the incubation chamber. Empty test tubes/cards are kept in the agitation block to maintain a pre-test temperature of 37°C.
• the agitation block is removed from the incubation chamber prior to the addition of test reactants.
• the test reactants i.e., red blood cells and plasma/serum
• a pre-labeled tube/card is also placed in a pre-drilled groove on the agitation block.
• the agitation block is then returned to the incubation chamber and is turned on to allow the mixing of the test reactants for a period of time, for example, from about 2 minutes, at the predetermined agitation rate, whether in a rotary or reciprocal agitation motion and in either case at a speed in accordance with that typically used by one having ordinary skill in the art of agitating such admixtures.
• the agitation block is removed from the incubation chamber. The test reactants are added to the tubes/card microtubes.
• the tube/card is placed in an appropriate centrifuge and centrifuged sufficient to advance any agglutinates formed to the bottom of the tube/microtube of the card.
• a six-tube agitation block design was selected to accommodate the six test tubes.
• the design of the agitation block can be configured to meet the requirements for other test technologies and automation platforms .
• Alternative agitation block designs can accommodate any other appropriate vessel for mixing purposes.
• alternative centrifuge designs can accommodate any other appropriate vessel for centrifugation purposes.
• a red blood cell diluent with a reduced ionic strength may be used to further enhance the rate of antibody uptake on the test cells.
• the MTS Diluent 2TM designed for antiglobulin testing in the ID-Micro Typing SystemTM Gel Test, was diluted with deionized water as described hereinbelow, in initial timed studies and evaluated for increased antibody uptake. The results of this study determined the optimal dilution of MTS Diluent 2TM for the test system.
• the low ionic strength red blood cell diluent's final formulation was designed with the following proportions: 7 ml of Deionized water was added to 10 ml of
• ID-MTS Diluent 2TM STAT Indirect Antiglobulin Procedure - Gel Test
• the indirect antiglobulin procedure that capitalizes on maximizing the test advantages of combining agitation and low ionic strength solutions uses the following testing protocol for a gel test application. All reagents and samples are brought to room temperature prior to use. Sample requirements include EDTA plasma. The screening cells are diluted to a concentration of 0.4% in the low ionic strength diluent prior to use.
• the agitation block containing the six preheated tubes is removed from the agitation- incubation device.
• An Anti-IgG ID-Micro Typing SystemTM gel card is labeled with appropriate patient/test information and is placed in the holder on the agitation block.
• the appropriate microtubes of the labeled card are aligned in front of their corresponding test tubes. Colored lines are embedded on the agitation block for alignment guides.
• a volume of the appropriate 0.4% screening cell is pipetted into a tube, followed by addition of the patient's EDTA plasma pipetted into screening cell tubes. Agitation power is turned on. Agitation proceeds for 2 minutes and automatically shuts down.
• the agitation block is removed from the incubator.
• a volume of test sample is added to the appropriate microtube card from the corresponding test tube.
• the card is centrifuged for 10 minutes in an MTS centrifuge (Micro Typing Systems, Inc., Pompano Beach, FL) .
• the ionic strength of the red blood cell diluent, incubation temperatures, and the agitation rate are variables in the above procedure and can be altered and still achieve a reduction of incubation time .
• time to result in emergency testing is shortened (i.e., STAT orders)
• the process is applicable to blood bank automation platforms
• the' time to result is shortened for both automation and manual testing
• the process is applicable to all existing blood bank technologies and formats: gel, tube, and solid phase.
• the appropriate microtubes of the labeled gel card were aligned in front of their corresponding test tubes. Colored lines were embedded on the agitation block for alignment guides. 200 ⁇ l of the appropriate 0.4% screening cell (as diluted above) was pipetted into a tube. 100 ⁇ l of the patient's EDTA plasma was then pipetted into screening cell tubes. Agitation power was turned on. Agitation proceeded for 2 minutes and automatically shut down. The agitation block was removed from the incubator. 150 ⁇ l of the test sample was added to the appropriate microtube on the gel card from the corresponding test tube. The card was centrifuged for 10 minutes at 900rpm in the MTS centrifuge.
• Part A The materials and procedures of Part A were repeated with the exception that the sample was not agitated. The time to reach maximum titer was 10 minutes.
• Part A The materials and procedures of Part A were repeated with the exception that the sample was incubated at room temperature (about 18-25°C) . The time to reach maximum titer was 15 minutes.
• Results are tabulated at Table 1.
• the Maximum Titer column is reported as "8" as maximum agglutination results were achieved with the 1:8 dilution of the sample.
• Part D Incubation at Room Temperature with No Agitation
• the materials and procedures of Part A were repeated with the exceptions that the sample was incubated at room temperature (about 18-25°C) , and that the sample was not agitated. The time to reach maximum titer was 20 minutes. Results are tabulated at Table 1. The Maximum Titer column is reported as "8" as maximum agglutination results were achieved with the 1:8 dilution of the sample. It will be noted that agitation decreased the time required for incubation from the standard ID-MTS procedure as contained on the package insert by 11 minutes at 37°C.
• All reagents and samples are brought to room temperature prior to use.
• the sample requirements include use of EDTA plasma.
• Screening cells are diluted to a concentration of 0.4% in the low ionic strength diluent, (which is 10 mL of the MTS Diluent 2TM further diluted in 7 ml of deionized water) .
• the agitation block containing the pre-heated ID-MTS Anti-IgG (Rabbit) Gel Card which is labeled with appropriate patient/test information, is removed from the agitation-incubation device and is placed in the holder on the agitation block. Colored lines are embedded on the agitation block for alignment guides.
• 200 ⁇ l of the appropriate 0.4% screening cell (as diluted above) is pipetted into a microtube of the gel card.
• 100 ⁇ l of the patient's EDTA plasma is then pipetted into the screening cell microtube.
• Agitation power is turned on. Agitation proceeds for 2 minutes and automatically shuts down.
• the agitation block is removed from the incubator.
• 150 ⁇ l of the test sample is added to the appropriate microtube on the gel card.
• the card is centrifuged for 10 minutes at 900 rpm in the MTS centrifuge. The agglutination result is then read.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Hematology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Cell Biology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Ultra Sonic Daignosis Equipment (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=34738673

Family Applications (1)

Country Status (6)

Families Citing this family (12)

Family Cites Families (14)

• 2004
  • 2004-12-17 WO PCT/US2004/042531 patent/WO2005064347A1/fr active IP Right Grant
  • 2004-12-17 DE DE602004012161T patent/DE602004012161T2/de active Active
  • 2004-12-17 EP EP04814682A patent/EP1709451B1/fr active Active
  • 2004-12-17 JP JP2006547177A patent/JP4884981B2/ja active Active
  • 2004-12-17 AT AT04814682T patent/ATE387630T1/de not_active IP Right Cessation
  • 2004-12-17 US US10/582,887 patent/US7767436B2/en active Active
• 2010
  • 2010-05-04 US US12/773,377 patent/US7943368B2/en active Active

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events